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Drimia Jacq., a bulbous geophyte belonging to family Hyacinthaceae Batsch ex Borkh. is well known for its medicinal values. Leaves and bulbs of eight genotypes of Drimia were sequentially extracted with three solvents (DCM, MeOH and H2O) and analysed for their phytochemicals (TPC, TFC and TTC) and in vitro antioxidant activities (DPPH, ABTS radical scavenging activities and phosphomolybdenum assay). Bulbs of D. indica (triploid form) extracted with MeOH showed the highest TPC (22.19 ± 1.00 mg GAE/g DW). Higher TFC was recorded in MeOH extract of bulbs of D. raogibikei (58.65 ± 1.95 mg QE/g DW) whereas significantly maximum TTC (857.17 ± 38.62 mg GAE/g DW) was found in MeOH extract of D. razii. DPPH radical scavenging activity was highest in DCM bulb extract of D. coromandeliana (12.22%). DCM extract of D. polyantha showed greatest (64.26%) ABTS radical scavenging activity. Phosphomolybdenum reduction potential was maximum in MeOH leaf extract of D. indica (triploid form) (52.86 ± 0.20 mg AAE/g DW). RP-HPLC estimation of cardiac glycoside (Scillaren A) from the bulbs of eight genotypes of Drimia revealed the highest (5.54 ± 0.03 mg/g DW) quantity in DCM extracts of D. coromandeliana. Presence of a bufadienolide (Scillaren A) was confirmed by FTIR and HR-LCMS. Additionally, HR-LCMS analysis revealed the presence of three cardenolides (convallatoxin, digoxigenin monodigitoxoside and peruvoside) and 160 major metabolites in the bulbs of Drimia species. Present study is the first comparative account of phytochemicals, antioxidants, cardiac glycosides and LC-MS profiling of major metabolites from eight Indian Drimia genotypes.

Background and Objective: Previously, it was demonstrated that marinobufagenin (MBG) is implicated in the development of ethanol withdrawal in rats. It has been shown that ethanol withdrawal is associated with a pressor response in the alcoholics. We hypothesized that elevated levels of sodium pump ligand, MBG, would underline the increase in systolic blood pressure during alcohol withdrawal in humans. Methods: The cohort included 9 patients with the diagnosis “alcohol dependence syndrome” (F10.(1-3) according to ICD-10). The blood samples for measurement of MBG concentration were collected from the subjects on the first day of withdrawal and after 7 days treatment of the abstinence. Arterial blood pressure was measured via plethysmography at the same time points. Results: The beginning of the alcoholic abstinence was associated with the rise of arterial blood pressure with enhanced levels of plasma MBG. At day 7 following withdrawal, the systolic blood pressure and MBG levels were decreased to normal values. Conclusion: The development of alcohol withdrawal is accompanied by an increase in arterial blood pressure, which is associated with increased plasma MBG concentration.

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

Cinobufagin, a bufadienolide of toad venom of Bufo bufo gargarizans, is used as a cardiotonic, central nervous system (CNS) respiratory agent, as well as an analgesic and anesthetic. However, several research showed that bufadienolide has a few side effects on the CNS, such as breathlessness or coma. Although cinobufagin was shown to display pharmacological effects in various models, the toxic effect of cinobufagin is elusive in brain cell models. The aim of this study was to explore whether cinobufagin affected viability, Ca2+ homeostasis, and reactive oxygen species (ROS) production in Gibco® Human Astrocyte (GHA) and HCN-2 neuronal cell line. In GHA cells but not in HCN-2 cells, cinobufagin (20-60 μM) induced [Ca2+ ]i rises. In terms of cell viability, chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid reduced cinobufagin-induced cytotoxicity in GHA cells. In GHA cells, cinobufagin-induced Ca2+ entry was inhibited by 2-aminoethoxydiphenyl borate or SKF96365. In a Ca2+ -free medium, treatment with thapsigargin or U73122 abolished cinobufagin-evoked [Ca2+ ]i rises. Furthermore, treatment with N-acetylcysteine reversed ROS production and cytotoxicity in cinobufagin-treated GHA cells. Together, in GHA cells but not in HCN-2 cells cinobufagin caused cytotoxicity that was linked to preceding [Ca2+ ]i rises by Ca2+ influx via store-operated Ca2+ entry and phospholipase C-dependent Ca2+ release from the endoplasmic reticulum. Moreover, cinobufagin induced ROS-associated cytotoxicity.

Background Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide amphibian biodiversity loss. Many amphibians exhibit skin-based chemical defences, which may play an important role against invading pathogens, but whether the synthesis of these chemical compounds is enhanced or suppressed in the presence of pathogens is largely unknown. Here we investigated direct and indirect effects of larval exposure to the globally distributed and highly virulent Bd-GPL strain on skin secreted chemical defences and life history traits during early ontogeny of agile frogs (Rana dalmatina) and common toads (Bufo bufo). Results Exposure to Bd during the larval stage did not result in enhanced synthesis of the antimicrobial peptide Brevinin-1 Da in R. dalmatina tadpoles or in increased production of bufadienolides in B. bufo tadpoles. However, exposure to Bd during the larval stage had a carry-over effect reaching beyond metamorphosis: both R. dalmatina and B. bufo froglets contained smaller quantities of defensive chemicals than their Bd-naïve conspecifics in the control treatment. Prevalence of Bd and infection intensities were very low in both larvae and metamorphs of R. dalmatina, while in B. bufo we observed high Bd prevalence and infection intensities, especially in metamorphs. At the same time, we did not find a significant effect of Bd-exposure on body mass or development rate in larvae or metamorphs in either species. Conclusions The lack of detrimental effect of Bd-exposure on life history traits, even parallel with high infection intensities in the case of B. bufo individuals, is surprising and suggests high tolerance of local populations of these two species against Bd. However, the lowered quantity of defensive chemicals may compromise antimicrobial and antipredatory defences of froglets, which may ultimately contribute to population declines also in the absence of conspicuous mass-mortality events.

1. Inducible defences are ubiquitous in the animal kingdom, but little is known about facultative changes in chemical defences in response to predators, especially so in vertebrates. 2. We tested for predator-induced changes in toxin production of larval common toads (Bufo bufo), which are known to synthesize bufadienolide compounds. 3. The experiment included larvae originating from three permanent and three temporary ponds reared in the presence or absence of chemical cues of three predators: dragonfly larvae, newts or fish. 4. Tadpoles raised with chemical cues of predation risk produced higher numbers of bufadienolide compounds and larger total bufadienolide quantities than predator-naive conspecifics. Further, the increase in intensity of chemical defence was greatest in response to fish, weakest to newts and intermediate to dragonfly larvae. Tadpoles originating from temporary and permanent ponds did not differ in their baseline toxin content or in the magnitude of their induced chemical responses. 5. These results provide the first compelling evidence for predator-induced changes in chemical defence of a vertebrate that may have evolved to enhance survival under predation risk.

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na+ /K+ -ATPase pump by inhibiting the cardiotonic steroid dependent-site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt-sensitive hypertensive states such as essential hypertension, congestive heart-failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin-layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid-phase extraction coupled with fractionation on high-performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid-phase extraction combined with high-performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time-consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.

Possessing toxins can contribute to an efficient defence against various threats in nature. However, we generally know little about the energy- and time-demands of developing toxicity in animals, which determines the efficiency of chemical defence and its trade-off with other risk-induced phenotypic responses. In this study we examined how immersion into norepinephrine solution inducing the release of stored toxins, administration of mild stress mimicking predator attack or simple handling during experimental procedure affected the quantity and number of toxin compounds present in common toad (Bufo bufo) tadpoles as compared to undisturbed control individuals, and investigated how fast toxin reserves were restored. We found that total bufadienolide quantity (TBQ) significantly decreased only in the norepinephrine treatment group immediately after treatment compared to the control, but this difference disappeared after 12 h; there were no consistent differences in TBQ between treatments at later samplings. Interestingly, in the norepinephrine treatment approximately half of the compounds characterized by >700 m/z values showed the same changes in time as TBQ, but several bufadienolides characterized by

The population of Bufo bufo gargarizans Cantor in China has been alarmingly declining due to environmental pollution. Deltamethrin is a pyrethroid pesticide frequently used in agriculture and much of its residues are present in crops, soil and water. Deltamethrin has been shown to have toxicity to toads. Herein, we assumed that deltamethrin contamination might influence the biosynthesis of toxic substances present in toad venom. Bufadienolides present in venom are the toad's chemical defense and highly toxic to predators, and they are important for the survival of toad species against predators. In this study, we determined the contents of bufadienolides in toad venom using a HPLC-triple quadrupole mass spectrometer to evaluate the change in bufadienolide profiles in toad venom before and after cutaneous exposure to deltamethrin. The results indicated that toads exposed to high concentration of deltamethrin survive the least, do not exuviate, and their movements are stiff. Furthermore, it was observed that high level of deltamethrin contamination induces a marked decrease in the levels of toxic bufadienolides in toad venom. These changes in the toxin profiles could lead to the compromised chemical defense of toad, leading to more susceptible to attack by predators. This is the first study to report that environmental contaminants (pesticides) can influence the toad's toxic profiles, suggesting one factor contributing to the decline in the population of B. bufo gargarizans Cantor.

The Japanese natricine snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads in the nuchal glands as defensive toxins. A previous study showed that R. tigrinus in captivity converts dietary BDs when it sequesters them. However, it is unknown whether the dietary BDs are actually converted and the modified products accumulated under natural conditions. It is also unknown to what extent the BD profile of ingested toads is reflected in that of the snake. We collected 123 snakes from throughout Japan, analyzed their BD profiles by liquid chromatography/mass spectrometry, and identified 15 BDs from R. tigrinus by nuclear magnetic resonance analyses. We also compared their BD profiles using hierarchical cluster analysis (HCA). HCA exhibited two main clusters associated with their collection locations: eastern and western regions of the Japanese main islands. These results, coupled with previous findings on the BDs of Japanese toads, suggest that 1) R. tigrinus converts toad-derived BDs into other compounds under natural conditions; 2) there are both universal and regionally-specific conversions of dietary BDs by R. tigrinus; and 3) geographic variation in toad BD profiles is partially reflected in the variation of snake BD profiles.

Toad venom, a traditional natural medicine, has been used for hundreds of years in China for treating different diseases. Many studies have been performed to elucidate the cardiotonic and analgesic activities of toad venom. Until the last decade, an increasing number of studies have documented that toad venom is a source of lead compound(s) for the development of potential cancer treatment drugs. Research has shown that toad venom contains 96 types of bufadienolide monomers and 23 types of indole alkaloids, such as bufalin, cinobufagin, arenobufagin, and resibufogenin, which exhibit a wide range of anticancer activities in vitro and, in particular, in vivo for a range of cancers. The main antitumor mechanisms are likely to be apoptosis or/and autophagy induction, cell cycle arrest, cell metastasis suppression, reversal of drug resistance, or growth inhibition of cancer cells. This review summarizes the chemical constituents of toad venom, analyzing their anticancer activities and molecular mechanisms for cancer treatments. We also outline the importance of further studies regarding the material basis and anticancer mechanisms of toad venom.

Bryophyllum pinnatum has been used in the treatment of premature labor, first in anthroposophic hospitals and, recently, in conventional settings as an add-on medication. In vitro work with hTERT human myometrial cells showed that B. pinnatum leaf press juice inhibits the increase of intracellular free calcium concentration induced by oxytocin, a hormone known to play a role in labor. Our aim was to identify fractions/compounds in B. pinnatum press juice that contribute to this inhibitory effect, and to investigate their effect on oxytocin-driven activation of the MAPK cascade. Several fractions/compounds from B. pinnatum press juice led to a concentration-dependent decrease of oxytocin-induced increase of intracellular free calcium concentration, but none of them was as strong as B. pinnatum press juice. However, the combination of a bufadienolide and a flavonoid-enriched fraction was as effective as B. pinnatum press juice, and their combination had a synergistic effect. B. pinnatum press juice inhibited oxytocin-driven activation of MAPKs SAPK/JNK and ERK1/2, an effect also exerted by the bufadienolide-enriched fraction. The effect of B. pinnatum press juice on oxytocin-induced signaling pathways was comparable to that of the oxytocin-receptor antagonist and tocolytic agent atosiban. Our findings further substantiate the use of B. pinnatum press juice preparations in the treatment of preterm labor.

Context Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. Objective The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate – a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. Materials and methods The cytotoxic activity of the compound (at 0.1–20.0 μg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. Results The compound had strong effect on the cells (IC50 = 0.55 μg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. Conclusions Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.

Kinetic properties and crystal structures of the Na+,K+-ATPase in complex with cardiotonic steroids (CTS) revealed significant differences between CTS subfamilies (Laursen et al., 2015): beneficial effects of K+ on bufadienolide binding strongly contrasted with K+/cardenolide antagonism. To solve this riddle we applied docking and molecular dynamics simulations of the complexes involving Na+,K+-ATPase, bufadienolides (bufalin, cinobufagin), and ions (K+, Na+, Mg2+). The results revealed that bufadienolide binding is affected by i) electrostatic attraction of the lactone ring by a cation, and ii) the ability of a cation to stabilize and “shape” the site constituted by transmembrane helices of the α-subunit (αM1-6). The latter effect was due to varying coordination patterns involving amino acid residues from helix bundles αM1-4 and αM5-10. Substituents on the steroid core of a bufadienolide add to and modify the cation effects. The above rationale is fully consistent with the ion effects on the kinetics of Na+,K+-ATPase/bufadienolide interactions.

A new bufadienolide (1), two new bufadienolide glycosides (2 and 3), a new ecdysteroid (4), and four known compounds (5–8), were isolated from the whole plants of Helleborus niger L. (Ranunculaceae). The structures of the new compounds (1–4) were determined by spectroscopic analysis, including 2D NMR spectral data, and hydrolytic studies. Compounds 1–6 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC50 values ranging from 0.0055 to 1.9 µM. HL-60 cells treated with either 3 or 4 showed apoptosis characteristics, such as nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3/7.

1. Toxin resistance in all taxa is important for their survival. Resistance to bufadienolide toxin is mediated by molecular changes to the sodium-potassium-pump. As for toads themselves, the parotid is consisted of extremely high concentration of bufadienolide toxin, it indicates toads need stronger resistance for self-protection than any other species involved in toad predation. Thus, besides the widespread convergence of Na+/K+-ATPase, toads may develop a unique self-resistance way for their accumulated bufadienolide toxin. 2. Our studies on invasive cane toads from Australia and common asiatic toads from China showed that toads store defensive venom in parotid as a combinatorial library composed of low-toxic protoxin (conjugated bufadienolides), which is characterized by two building blocks, i.e. high-toxic toxin (free bufadienolide) and the argininyl side chain contributed to toxin absorption promotion. This protoxin could be hydrolyzed to high-toxic toxin for defense immediately as it is discharged from parotid at the time when toad is attacked or stimulated by predators. 3. Our results revealed that toads indeed develop a unique protoxin self-resistance way to their own extremely high concentration of bufadienolide toxin for self-protection besides the widespread convergence of Na+/K+-ATPase. The revelation of this protoxin self-resistance way highlights the importance and ecological multifunctionality of the sophisticated combinatorial chemistry of nature and may partly account for the strong adaptability of toad as infamous ailing invaders. It completes the picture of self-resistance mechanism to their own bufadienolide toxin for toad or may even for many other venomous animals.

This study presents an efficient strategy based on liquid-liquid extraction and pH-zone-refining counter-current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two-phase solvent system with maximum or minimum partition coefficient was developed for the liquid-liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH-zone-refining counter-current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin-3-adipoyl-l-arginine, 42 mg of telocinobufagin-3-pimeloyl-l-arginine, 51 mg of telocinobufagin-3-suberoyl-l-arginine, 132 mg of marinobufagin-3-suberoyl-l-arginine, and 57 mg of bufalin-3-suberoyl-l-arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.

Bufadienolides are cardioactive C24 steroids with an α-pyrone ring at position C17. In the last ten years, accumulating studies have revealed the anticancer activities of bufadienolides and their underlying mechanisms, such as induction of autophagy and apoptosis, cell cycle disruption, inhibition of angiogenesis, epithelial-mesenchymal transition (EMT) and stemness, and multidrug resistance reversal. As Na+/K+-ATPase inhibitors, bufadienolides have inevitable cardiotoxicity. Short half-lives, poor stability, low plasma concentration and oral bioavailability in vivo are obstacles for their applications as drugs. To improve the drug potency of bufadienolides and reduce their side effects, prodrug strategies and drug delivery systems such as liposomes and nanoparticles have been applied. Therefore, systematic and recapitulated information about the antitumor activity of bufadienolides, with special emphasis on the molecular or cellular mechanisms, prodrug strategies and drug delivery systems, is of high interest. Here, we systematically review the anticancer effects of bufadienolides and the molecular or cellular mechanisms of action. Research advancements regarding bufadienolide prodrugs and their tumor-targeting delivery strategies are critically summarized. This work highlights recent scientific advances regarding bufadienolides as effective anticancer agents from 2011 to 2019, which will help researchers to understand the molecular pathways involving bufadienolides, resulting in a selective and safe new lead compound or therapeutic strategy with improved therapeutic applications of bufadienolides for cancer therapy.

Kalanchoe species are well-known medicinal plants used in traditional medicine as anti-inflammatory and analgesic remedies. Recently, it has been reported that Kalanchoe plants have cytotoxic properties; however, data on traditional use of these plants in tumor treatment are extremely limited. Kalanchoe daigremontiana is one of the most popular species cultivated in Europe, and it is used, among other things, as a remedy in treating skin injuries and wounds. Studies on the biological activity of this species are scarce, and there is a lack of data on the cytotoxic activity of K. daigremontiana extracts on epithelial cancer cells in the literature. In our present study, we analyzed the phytochemical composition of K. daigremontiana ethanol extract and fractions–water and dichloromethane–by the HPLC-DAD-ESI-MS method and estimated cytotoxic activity of the extracts on human adenocarcinoma (HeLa), ovarian (SKOV-3), breast (MCF-7), and melanoma (A375) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, real-time cell analyzer (RTCA), and flow cytometry. We identified 6 bufadienolide compounds and 19 flavonoids, mostly kaempferol, quercetin, isorhamnetin, and myricetin glycosides, of which only 3 flavonoids have been identified in K. daigremontiana to date. Other flavonoids that were characterized in our study have not yet been found in this plant. The ethanol extract and water fraction of K. daigremontiana did not show significant cytotoxic activity on the tested cell lines. In contrast, the dichloromethane fraction showed the strongest activity against all cell lines with IC50 values of ≤ 10 µg/mL. The results indicated that this activity is mainly due to the presence of bersaldegenin-1,3,5-orthoacetate.

Cytotoxicity-guided fractionation of the MeOH extract of Helleborus lividus Aiton ex Curtis (Ranunculaceae) resulted in the isolation of five undescribed bufadienolide glycosides and two undescribed bufadienolides, along with three known compounds. Their structures were determined by detailed spectroscopic analysis and hydrolysis studies. The isolated compounds showed cytotoxicity against HL-60 human leukemia cells and A549 human lung adenocarcinoma cells, with IC50 values ranging from 2.20 ± 0.01 nM to 0.77 ± 0.01 μM. The undescribed compound 3β-[(O-β- d -glucopyranosyl-(1 → 4)-α- l -rhamnopyranosyl)oxy]-14β,16β-dihydroxy-5β-bufa-20,22-dienolide induced apoptosis in HL-60 cells via a mitochondria-dependent apoptotic pathway. The average IC50 values of bufadienolide monorhamnosides for HL-60 and A549 cells were 10–20 times lower than those for Na+/K+ ATPase, implying that they induce tumor cell death via a mechanism of action other than Na+/K+ ATPase inhibition.

Bryophyllum pinnatum has been used since the 1970s to prevent premature labour, first in anthroposophic hospitals and, more recently, also in the main Swiss perinatal centres. However, it is not known which compounds in B. pinnatum leaves contribute to the tocolytic effect. Here we studied the effects of a flavonoid-enriched fraction, the corresponding flavonoid aglycon mixture, a bufadienolide-enriched fraction, and B. pinnatum leaf press juice on human myometrial contractility in vitro. The strength (area under the curve and amplitude) and frequency of contractions were recorded using strips of human myometrium mounted in an organ bath system. Cell viability assays were performed with the human myometrium hTERT-C3 and PHM1 – 41 cell lines. Repeated addition of the flavonoid-enriched fraction, flavonoid aglycon mixture, bufadienolide-enriched fraction, or B. pinnatum leaf press juice led to a progressive decrease of contraction strength, without jeopardising the vitality of myometrium strips. The bufadienolide-enriched fraction was the most active, since 1 µg/mL of the bufadienolide-enriched fraction lowered the area under the curve to 40.1 ± 11.8% of the initial value, whereas 150 µg/mL of the flavonoid-enriched fraction, 6.2 µg/mL of the flavonoid aglycon mixture, and 10 mg/mL of the B. pinnatum leaf press juice were required to achieve comparable inhibition. A progressive increase of contraction frequency was observed, except in the case of the flavonoid aglycon mixture, which did not affect frequency. None of the test substances decreased myometrial cell viability, even at concentrations of 500 µg/mL of the flavonoid-enriched fraction, 40 µg/mL of the flavonoid aglycon mixture, 3.8 µg/mL of the bufadienolide-enriched fraction, and 75 mg/mL of the B. pinnatum leaf press juice, i.e., higher than those used in the myometrium experiments. Given the concentrations of flavonoids in the flavonoid-enriched fraction and B. pinnatum leaf press juice, and of bufadienolides in the bufadienolide-enriched fraction and B. pinnatum leaf press juice, it appears that bufadienolides may be mainly responsible for the relaxant effect.

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

In many amphibians, the granular glands can be grouped in special regions forming macroglands. This is the case of toads, characterized by the presence of a pair of parotoid macroglands, strategically located to give protection by poison release in case of attacks. The product secreted consists of a wide variety of chemical compounds including proteins, peptides, biogenic amines, toxic steroidal bufadienolides, and various alkaloids, depending on the species. In this work, using Rhinella arenarum, we have performed, for the first time, the matrix assisted-ultraviolet laser desorption/ionization mass spectrometry and tandem mass spectrometry characterization of the components of the secretion used as crude material, just suspended in MeOH (or MeCN). The crude sample as a whole (whole suspension) was spotted on the matrix assisted-ultraviolet laser desorption plate for analysis. Electrospray ionization-Orbitrap was used for cross-checking experiments. The pattern of signals obtained at m/z ranges 600 to 800 and 1200 to 1600 could be assigned as the argininyl bufadienolide esters fingerprint characteristic of female and male. Variation patterns for gender (female, male), age (non-reproductive, reproductive), and season (non-reproductive, reproductive) are described. Fil: Petroselli, Gabriela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Raices, Marilina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Jungblut, Lucas David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Pozzi, Andrea Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Erra Balsells, Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina

Objective Kalanchoe hybrida (Crassulaceae) is naturalized throughout all the island of Taiwan in China. The preliminary bioassay-guided fractionation of the crude extract of K. hybrida exhibited that the chloroform and n-butanol fractions possessed potent cytotoxicity against MCF-7, NCI-H460, and SF-268 tumor cell lines at 50 µg/mL concentration. Therefore, K. hybrida was selected as a target and the chemical constituents from the chloroform and n-butanol fractions of the crude extracts of K. hybrida were identified. The potential constituents were examined for their cytotoxicity against the tumor cell lines. Methods A combination of conventional chromatographic techniques was performed on the crude extract of K. hybrida. The chemical structures of the purified constituents were identified on the basis of spectroscopic and spectrometric analysis. Results The purification results had led to the characterization of totally 37 compounds. The isolated compounds 1, 2, and 4–12 were examined for their cytotoxicity in vitro, and bufadienolides 4–8 and flavonol glycoside 11 displayed significant cytotoxicity towards all the tested tumor cell lines among these tested compounds. Conclusion The results indicated that these principles should be responsible for the bioactivity of corresponding partial fractions. The potential constituents could be further investigated to explore the new natural lead drugs.

Two new bufadienolides (1 and 2) and three known bufadienolide glucosides (3–5) were isolated from the whole plants of Helleborus foetidus (Ranunculaceae). The structures of 1 and 2 were determined spectroscopically. Compound 1 displayed potent cytotoxicity against HL-60 and A549 cells, with IC50 values of 0.035 and 0.029 μM, respectively. HL-60 cells treated with 1displayed typical characteristics of apoptosis, such as nuclear chromatin condensation, DNA fragmentation, accumulation of sub-G1 cells, and marked activation of caspase-3. Moreover, the loss of mitochondrial membrane potential and release of cytochrome c into the cytosol suggested that 1 induced HL-60 cell death via the mitochondrial-dependent apoptotic pathway.

Bufadienolides are a class of natural cardiotonic steroids and are well known for their antitumor activities. Their clinical use has been hindered by poor water solubility. Enzymatic glycosylation is a favorable approach to improve the solubility of natural products. In this work, we describe the highly efficient one-step synthesis of bufadienolide O-glucosides and O-galactosides using a microbial glycosyltransferase YjiC1, with conversion rates from 63% to 99%. Altogether 24 glycosides including 17 new compounds were obtained from 14 substrates, and their structures were identified by extensive NMR and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses. These products belong to five types: glycosylation at 3 beta-OH, 7 beta-OH, 3 beta,7 beta-di-OH, 11 alpha-OH, and 12 beta-OH. The C-7 beta or C-11 alpha O-glycosides of bufadienolide are reported for the first time. Moreover, the 3-O-glycosides exhibited significant cytotoxic activities against A549 and MCF7 human cancer cell lines, and showed potent inhibitory activities against Na+/K+-ATPase with IC50 values in the range 0.053-0.76 mu M. In particular, bufalin 3-O-beta-D-glucoside showed enhanced water solubility (25-fold increase from bufalin) and potent antitumor activities (30% inhibition rate, 1.4 mg kg(-1), i.p.) on A549 human lung cancer xenograft Balb/c nude mice model, and could be a promising drug candidate.

Objectives: This study aimed to predict the binding pose of bufadienolides from Kalanchoe pinnata on Na+/K+-ATPase using molecular docking in comparison with a known inhibitor, and to determine the important functional group of bufadienolide that is responsible for good binding. Material and methods: Docking was performed on the receptor file (PDB ID: 4RES) using AutoDock 4.2. Before docking, the complex structure of ligand-receptor was minimized using AMBER 14. The physico-chemical properties were predicted using Biovia Draw. Results: The sterical hindrance of Glu117 to the 1,3,5-orthoacetate moiety was removed after minimisation. The docking energy, Polar Surface Area (PSA) and A Log P showed good correlation with the experimental data. The 14-OH of all bufadienolides formed a hydrogen bond with Thr797. However, the energy breakdown analysis of docking result demonstrated that C1 has the highest ligand efficiency with the strongest hydrogen bond and electrostatic energies, followed by C2 and C3. The hydrogen bond from the 10-OH and the orthoacetate ring in C1 was stronger than that of 10-CH2OH in C2 and 10-OH in C3. Conclusion: The best activity of C1, as compared to C2 and C3, was due to the presence of 1,3,5-orthoacetate moiety, 10-CHO, 11-OH, and 14-OH. In addition, the 11-OH group appeared to decrease the toxicity of the bufadienolide by improving its selectivity to the receptor. This result is expected to be useful in the further development of bufadienolide-based inhibitor for anticancer. Key words: Bufadienolide, Kalanchoe Pinnata , Anticancer, Na+/K+-Atpase, Molecular Docking.

Many animals display spectacular adjustments of their phenotype to changing environmental conditions in terms of altered behaviour, physiology and morphology, but whether similar plasticity in chemical defences is taxonomically widespread as well has remained unknown. Bufadienolides, toxins produced by some anuran amphibians, are known to be effective against many predators, but they can also contribute to defences against microorganisms. We experimentally investigated if qualitative and/or quantitative changes in the chemical defences of Bufo bufo tadpoles are induced by differences in the microbiota of their environment. We reared animals in purified tap water, or in pond water containing lowered or natural quantities of the natural microbiota, and measured the number of compounds and total quantity of bufadienolides at three ontogenetic stages during larval development, as well as 10 days after metamorphosis. We observed a significant decrease in the number of compounds, and a parallel increase in the total quantity of bufadienolides after metamorphosis, independent from treatments. The quantity of individual bufadienolide compounds also changed during ontogeny independently from treatments. Our results, thus, suggest that although bufadienolide production changes during early ontogeny, it is not adjusted to the microbiota present in the environment.

Toad venom is regarded as the main source of bufadienolides; however, synthesis of these substances takes also place in a variety of other animal and plant organisms, including ethnomedicinal plants of the Kalanchoe genus. Chemically, bufadienolides are a group of polyhydroxy C-24 steroids and their glycosides, containing a six-membered lactone (α-pyrone) ring at the C-17β position. From the pharmacological point of view, bufadienolides might be a promising group of steroid hormones with cardioactive properties and anticancer activity. Most of the literature concerns bufadienolides of animal origin; however, the medicinal use of these compounds remains limited by their narrow therapeutic index and the risk of development of cardiotoxic effects. On the other hand, plants such as Kalanchoe are also a source of bufadienolides. Kalanchoe pinnata (life plant, air plant, cathedral bells), Kalanchoe daigremontiana (mother of thousands) and other Kalanchoe species are valuable herbs in traditional medicine of Asia and Africa. The present review focuses on the available data on chemical structures of 31 compounds, biological properties and prospects for therapeutic use of bufadienolides from Kalanchoe species. Furthermore, it presents some new investigational trends in research on curative uses of these substances.

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides-the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin-from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.

Thrombin is an active plasma coagulation factor II, critical for the formation of fibrin clot during blood coagulation. For that reason, this protein is also a crucial target for different anti-thrombotic therapies. The work is based on in vitro evaluation of the inhibitory effect of bufadienolide-rich fraction, isolated from roots of Kalanchoe daigremontiana (1-50μg/ml) on enzymatic properties of a serine proteinase - thrombin. The efficacy of the inhibition of amidolytic activity of thrombin (measured as a hydrolysis of the chromogenic substrate S-2238, Chromogenix) attained about 10 and 66%, respectively. The IC50, established for the examined bufadienolide fraction was 2.79μg/ml, while the IC50 calculated for argatroban (reference compound) was 0.78μg/ml. Linearization conducted using Lineweaver-Burk plot indicated that the K. daigremontiana fraction contains compounds that are uncompetitive inhibitors of thrombin. K. daigremontiana fraction was also able to reduce the proteolytic activity of thrombin towards its physiological substrate, i.e. fibrinogen. Additionally, this study is supported by in silico analysis of interactions of the most common compounds, identified in the examined in Kalanchoe extract to crystal structure of this enzyme.

The acute respiratory distress syndrome (ARDS) represents a major challenge for clinicians as well as basic scientists. The mortality rate for ARDS has been maintained within the range of 40-52%. The authors have examined the involvement of the "cardiotonic steroids" in the pathogenesis and therapy of ARDS. We have studied the possible role of the bufadienolide, marinobufagenin (MBG), in the pathogenesis of ARDS in both a rat model of ARDS and in patients afflicted with that disorder. In addition, the potential therapeutic benefit of an antagonist of MBG, resibufogenin (RBG), in an animal model has been evaluated.A syndrome resembling human ARDS was produced in the rat by exposing the animals to 100% oxygen for 48 h. In other animals, RBG was administered to these "hyperoxic" rats, and the serum MBG was measured. In human ICU patients, urinary samples were examined for levels of MBG, and the values were compared to those obtained from other ICU patients admitted with diagnoses other than ARDS.(1) Exposure of rats to hyperoxia produced a histologic picture which resembled that of human ARDS. (2) Serum levels of MBG in the "hyperoxic" rats substantially exceeded those obtained in animals exposed to ambient oxygen levels and were reduced to normal by RBG. (3) In ARDS patients, substantial elevations in urinary MBG were obtained compared to those in non-ARDS ICU patients.MBG may serve as an important biomarker for the development of ARDS, and RBG may represent a preventative/therapy in this disorder.

Bufospirostenin A (1) and bufogargarizin C (2), two novel steroids with rearranged A/B rings, were isolated from the toad Bufo bufo gargarizans. Compound 1 represents the first spirostanol found in animals. Compound 2 is an unusual bufadienolide with a cycloheptatriene B ring. Their structures were elucidated by spectroscopic analysis, single crystal X-ray diffraction analysis, and computational calculations.

Three pairs of bufadienolide l/d-lactate epimers (1-6) were isolated from the eggs of the toad Bufo bufo gargarizans. The structures were elucidated by using spectroscopic methods, X-ray diffraction analysis and a modified Mosher's method. Compounds 1-6 represent the first occurrence of lactate-conjugated bufadienolides in nature, and illustrate the existence of an enzyme-controlled epimerization from l- to d-lactate in amphibians. The biosynthetic pathways, in which two key enzymes might be involved (i.e., lactate racemase and acyltransferase), were proposed. In addition, the biological assays revealed that compounds 1-4 are potent cytotoxic agents against human gastric cancer cells BGC-823 and human lung cancer cells A549 with IC50 values in a range of 8.0 to 80.0 nM.

American trypanosomiasis is a parasitic neglected disease, responsible for the death of approximately 10,000 people every year. Amphibians are recognized for producing in their cutaneous glands substances with pharmacological potential against a variety of pathologies. Here we investigated the antiprotozoal activity against Trypanosoma cruzi of bufadienolides isolated from the parotoid glands secretions of the toad Rhinella centralis from Panama. NMR and mass spectrometry analysis led to the identification of the active compound 19-hydroxy-bufalin, for which its antitrypanosomal activity and occurrence in the genus Rhinella are reported for the first time. This compound showed low cytotoxicity and significant selectivity which confers to it a potential role for the treatment of Chagas disease.

Bufadienolides are a type of natural cardiac steroids and originally isolated from the Traditional Chinese Medicine Chan'Su, they have been used for the treatment of heart disease in traditional remedies as well as in modern medicinal therapy with potent anti-tumor activities. Due to their unique molecular structures with unsaturated six-membered lactones attached to the steroid core, bufadienolides have received great attention in the synthetic organic community. This review presents total synthetic efforts to some representative bufadienolides, chemical modification of bufadienolides will also be given to discuss their structure-activity relationship in anti-tumor.

Two undescribed bufadienolide glucosides and four undescribed bufadienolides were isolated from the whole plants of Helleborus foetidus (Ranunculaceae). Their structures were determined by extensive spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds exhibited cytotoxic activities against HL-60 and A549 cells with IC50 values ranging from 0.019 to 3.0 μM. The isolated compounds also showed the Na+/K+ ATPase inhibitory activity. The undescribed compound 16β-formyloxy-10,14-dihydroxy-5β-[(β- d -glucopyranosyl)oxy]-19-norbufa-3,20,22-trienolide induced apoptosis in HL-60 cells through a mitochondria-dependent apoptotic pathway.

Little is known on the composition of Peruvian Amazon toad venoms. The large toad Rhinella marina is common in the cleared tropical forests of the Iquitos region and is regarded as poisonous. The venom from two different populations of R. marina was collected in the Departamento de Loreto, Peru. The samples were assessed for antiproliferative effect and composition. Some 29 compounds were identified or tentatively identified from the venom by spectroscopic and spectrometric means. The main free bufadienolide was marinobufagin 7 while marinobufotoxin 15 and bufalitoxin 9 were the main bufadienolide argininyl diacid derivatives. The alkaloids dehydrobufotenin 28 and bufotenidin 29 were present in both venoms. The main difference in the venoms was the relative ratio of argininyl diacids from bufadienolides to free bufadienolides. The argininyl diacids included derivatives from bufalin, marinobufagin, telocinobufagin, hellebrigenin, resibufogenin and bufotalinin. Four compounds, including undecadienoyl aginine 6 and three argininyl diacids from bufadienolides were tentatively identified for the first time in the samples. The venom showed a strong antiproliferative effect towards MRC-5 normal human lung fibroblasts (0.063–0.247 μg/mL), AGS human gastric adenocarcinoma cells (0.076–0.272 μg/mL), SK-MES-1 human lung cancer cells (0.154–0.296 μg/mL), J82 human bladder carcinoma cells (0.169–0.212 μg/mL), and HL-60 human promyelocytic leukemia (0.071–0.283 μg/mL). The antiproliferative effect is mediated by ROS production and cell cycle arrest in human breast cancer cells (MCF7 and MDA-MB-231). This is the first report on the composition of R. marina venom from the Peruvian Amazon pointing out the need to include different venom samples to get a better picture from the activity and composition of South American toad defense substances.

Defensive chemicals in anuran skin secretions function in protection against potential predators. Although studies have demonstrated that particular chemicals are effective against certain predators, very little is known about how different chemicals from different species function against the same predators. Understanding how different chemicals function as a defence against similar predators is fundamental to the ecology and evolution of chemical defences in frogs. In the present study, the defensive function of bufadienolide-based defences in adultRhaebo haematiticus(Bufonidae) were compared with alkaloid-based defences in adult and juvenileDendrobates auratus(Dendrobatidae) against the same predators. Most bufonids contain synthesized bufadienolides, whereas dendrobatids contain dietary-derived alkaloids. Predation trials were performed with two potential invertebrate predators,Paraponera clavata(bullet ant) andCupiennius coccineus(ctenid spider), to determine how these predators respond to two different types of frog chemical defence. The non-chemically defended frogCraugastor fitzingeriserved as a control in all predation trials. Our results suggest that bufadienolide defences ofR. haematiticusand alkaloid defences ofD. auratusare equally effective towards bullet ant and ctenid spider predators. The similar avoidance and cleaning behaviours exhibited by these ants and spiders after contact with bufadienolides and alkaloids suggest that both types of defence are unpalatable to these arthropod predators.

Bufadienolides are a major class of bioactive compounds derived from amphibian skin secretion. Recent studies demonstrate that bufadienolides have a promising role in targeted cancer chemotherapy. However, extensive metabolism and inactivation strongly restrict the clinical applications of bufadienolides. This study aimed to systematically characterize the sulfation of six representative bufadienolides (including bufalin, resibufogenin, cinobufagin, bufotalin, telocinobufagin and deacetylcinobufagin) in amphibian skin secretion and to provide insight into the structure–sulfation relationship by experimentation and molecular docking analysis with series of bufadienolides and derivatives. For all the six representative bufadienolides, one corresponding monosulfate was detected in the incubation mixtures. The sulfates were accurately identified as bufadienolides 3-O-sulfates by NMR and HPLC-MSn techniques. Reaction phenotyping studies using human recombinant sulfotransferase (SULT) and liver S9 demonstrated that SULT2A1 mediated the formation of bufadienolide 3-O-sulfate with a high specific selectivity. Further kinetic evaluation demonstrated that deacetylcinobufagin could be used as a preferred probe of SULT2A1. The regio- and stereo-selective sulfation properties of SULT2A1 and the structural variation effects of bufadienolides were investigated by docking analysis, which revealed the significance of appropriate molecule orientation and hydrophobic interactions of motifs with SULT2A1 His99 residues. Additionally, significant differences between humans and animal species were observed in the sulfation of bufalin and resibufogenin. This study provided important data for elucidating the mechanisms of bufadienolide sulfation and leads to a better understanding of the bufadienolide–SULT interaction which can be further used in preclinical development and rational use of bufadienolides.

Two new 19-norbufadienolides (1 and 2), one new 14,15-epoxy bufadienolide (3), and eight rare bufadienolide–fatty acid conjugates (4–11), together with four known ones (12–15) were isolated from the eggs of toad Bufo bufo gargarizans. Their chemical structures were elucidated by extensive spectroscopic methods in combination with X-ray diffraction analyses. Furthermore, we tested the inhibitory effect of these compounds against breast cancer cell lines MCF-7 and MDA-MB-231. Most of them showed strong cytotoxicity with IC50 values less than 0.1 μM. The further mechanistic study showed that they could induce apoptosis and cycle arrest in the G2/M phase in MCF-7 cells.

Natural cardiac-active principles built upon the 14,16β-dihydroxy-5β,14β-androstane core and bearing a heterocyclic substituent at 17β, in particular, a cardenolide - oleandrin and a bufadienolide - bufotalin, are receiving a great deal of attention as potential anticancer drugs. The densely substituted and sterically shielded ring D is the particular structural feature of these compounds. The first synthesis of oleandrigenin from easily available steroid starting material is reported here. Furthermore, selected 17β-(4-butenolidyl)- and 17β-(3-furyl)-14,16β-dihydroxy-androstane derivatives were en route synthesized and examined for their Na+/K+-ATP-ase inhibitory properties as well as cytotoxic activities in normal and cancer cell lines. It was found that the furyl-analogue of oleandrigenin/bufatalin (7) and some related 17-(3-furyl)- derivatives (19, 21) show remarkably high Na+/K+-ATP-ase inhibitory activity as well as significant cytotoxicity in vitro. In addition, oleandrigenin 2 compared to derivatives 21 and 25 induced strong apoptosis in human cervical carcinoma HeLa cells after 24 h of treatment.

Ethnopharmacological relevance Bufo gargarizans (Cantor, 1842) (BGC), a traditional medicinal animal distributed in many provinces of China, is well known for the pharmaceutical value of Chansu and Chanpi. As traditional Chinese medicines (TCMs), Chansu and Chanpi, with their broad-spectrum of therapeutic applications, have long been applied to detoxification, anti-inflammation, analgesia, etc. Overarching objective We critically analyzed the current evidence for the traditional uses, chemical profiles, pharmacological activity, toxicity and quality control of BGC (Bufonidae family) to provide a scientific basis for future in-depth studies and perspectives for the discovery of potential drug candidates. Methodology All of the available information on active constituents and TCMs derived from BGC was obtained using the keywords “Bufo gargarizans”, “Chansu”, “Chanpi”, “Huachansu”, or “Cinobufacini” through different electronic databases, including PubMed, Web of Science, Chinese National Knowledge Infrastructure (CNKI), the Wanfang Database, and Pharmacopoeia of China. In addition, Chinese medicine books from different times were used to elucidate the traditional uses of BGC. Electronic databases, including the “IUCN Red List of Threatened Species”, “American Museum of Natural History” and “AmphibiaWeb Species Lists”, were used to validate the scientific name of BGC. Results To date, about 118 bufadienolide monomers and 11 indole alkaloids have been identified from BGC in total. The extracts and isolated compounds exhibit a wide range of in vitro and in vivo pharmacological effects. The literature search demonstrated that the ethnomedicinal uses of BGC, such as detoxification, anti-inflammation and the ability to reduce swelling and pain associated with infections, are correlated with its modern pharmacological activities, including antitumor, immunomodulation and attenuation of cancer-derived pain. Bufadienolides and indole alkaloids have been regarded as the main active substances in BGC, among which bufadienolides have significant antitumor activity. Furthermore, the cardiotoxicity of bufadienolides was discussed, and the main molecular mechanism involves in the inhibition of Na+/K+-ATPase. Besides, with the development of modern analytical techniques, the quality control methods of BGC-derived TCMs are being improved constantly. Conclusions An increasing number of reports suggest that BGC can be regarded as an excellent source for exploring the potential antitumor constituents. However, the future antitumor research of BGC needs to follow the standard pharmacology guidelines, so as to provide comprehensive pharmacological information and aid the reproducibility of the data. Besides, to ensure the efficacy and safety of BGC-derived TCMs, it is vital to construct a comprehensive quality evaluation model on the basis of clarifying pharmacodynamic-related and toxicity-related compositions.

Intoxication by Moraea pallida Bak. (yellow tulp) in livestock is of great importance in South Africa, ranking top among all plant-induced cardiac glycoside toxicosis. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. Treatment of poisoning is challenging and affected livestock often succumbs due to the stress of handling. Manipulating animals to resist poisoning is a potential management strategy. The goal of this study was to explore the potential to develop a vaccine against epoxyscillirosidine by raising antibodies against epoxyscillirosidine in sheep and to assess the neutralization ability of the antibodies in vitro. Epoxyscillirosidine was successfully conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA) rendering them immunogenic. The sheep, vaccinated with epoxyscillirosidine-KLH conjugate (n = 4) and KLH (n = 2) with Montanide, developed antibodies as determined with an indirect enzyme linked immunosorbent assay (ELISA). Total immunoglobulins from sera of vaccinated and control sheep that were purified and concentrated using ammonium sulphate precipitation were 11,940 and 7850 μg, respectively. The in vitro neutralization assay using the methyl blue tetrazolium bromide (MTT) cell viability assay indicated no significant difference (p > 0.05) between anti-epoxyscillirosidine-KLH and KLH antibodies. Rather, the antibodies seemed to enhance the cytotoxicity of epoxyscillirosidine in H9c2 cells. Thus, it is necessary to develop improved vaccination methods to generate antibodies capable of neutralizing the functional group responsible for epoxyscillirosidine toxicity.

Bufadienolides are the main bioactive components of Venenum Bufonis (VB) and have been widely used to treat different types of human cancers for decades. The bufadienolide content in VB varies significantly in materials from different geographical origins. In this work, a new strategy for the quality assessment of VB was developed through quantitative analysis of multi-components by single marker (QAMS). Cinobufagin was selected as the internal reference substance; seven bufadienolides were separated and simultaneously determined based on relative correction factors. The correlation coefficient value (r ≥ 0.9936) between QAMS and the normal external standard method proved the consistency of the two methods. According to the outcomes of 30 batches of VB samples, the contents of the seven bufadienolides were used for further chemometric analysis. All of the samples of VB from various geographical origins were divided into three categories based on hierarchical cluster analysis and radar plot, which indicated the crucial influence of geographical origins on VB. This study showed that QAMS combined with chemometristry could be used to comprehensively evaluate and effectively control the quality of VB from different geographical origins.

Two new 19-norbufadienolides (1 and 2) and one new 14,15-epoxy bufadienolide (3) alongside 16 known bufadienolides (4-19) were isolated from Bufonis Venenum that originated from the skin and parotid venom glands of an Asiatic toad (Bufo bufo gargarizans Cantor). The structures of these bufadienolides were elucidated based on the interpretation of their HRESIMS and NMR data. Compound 1, which had a unique peroxide, was established through extensive single-crystal X-ray diffraction. The two 19-norbufadienolides exhibited more potent cardiotonic activity in the isolated toad heart model and lower cytotoxicity against U87, U251, and LN-18 cell lines than other bufadienolides, such as bufalin and bufotalin. The results suggested that 19-norbufadienolides might be more suitable for developing cardiotonic agents with low cytotoxicity.

Drimia genus includes plants that used from ancient time for various ailments such as dropsy, respiratory ailment, bone and joint complications, skin disorders, epilepsy and cancer. Toxic properties of some Drimia species also were noted by ancient scientists and these plants have been traditionally used for rat control. Bufadienolides have been identified as the main constituents in the genus of Drimia. Phenolics, sterols, protein and some of other phytochemicals have been also isolated from these plants. Pharmacological and clinical studies have strongly approved their effect on cardiovascular system. Extracts and compounds isolated from Drimia species showed biological activities such as antibacterial, antifungal, antiviral, antioxidant, anti-inflammatory and insecticidal effects through several in vivo and in vitro studies. Moreover, cytotoxic and antitumor activities which may be related to bufadienolide content of these plants have been considered by many researchers. Traditional therapeutic values of these plants for treating respiratory and rheumatic ailments as well as skin disorders are needed to be validated through more researches. Toxic effects of these plants and isolated compounds have been investigated through several in vivo studies. Drimia plants and their isolated compounds have narrow therapeutic index, so patients should be prohibited from applying these plants without medical supervision and should be informed about the main intoxication symptoms before starting treatment. Moreover, interaction of Drimia plants with other constituents of traditional herbal mixtures as well as chemical and biological modalities for reducing toxicity of bufadienolide compounds can be subjected for future studies.

Objective: The aim of this study was to evaluate the chemical composition as well as to assess the cytotoxicity of Kalanchoe mortagei. Materials and Methods: The plant chemical composition was determined by HPLC coupled to mass spectrometer. The cytotoxicity of methanol extract from leaves of Kalanchoe mortagei (KB) was carried out against human cervical cancer cells (HeLa), breast cancer (MCF-7), brain cancer (U373), breast adenocarcinoma (MDA-MB-231) and umbilical vein endothelial cells (HUVEC) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. KB was added at various concentrations of 6.25, 12.5, 25, 50, 100 and 200 µg/ml. In addtion, cell morphological changes and apoptosis were also evaluated. Results: A bufadienolides-rich fraction (BRF) containing nine bufadienolides was identified by HPLC/MS which to significantly reduce proliferation of HeLa cells. Bufadienolides identified included 3β-Acetate Hellebrigenin, Bufotalinin, 19-Oxo-bufalin, Hellebrigenin, Monohydroxy-bufotalin, 19-oxo-desacetylcinobufagin, 1β-Hidroxybufalin, Bufalin, and 3-Dehydroxy cinobufagin. Discussion & Conclusion: K. mortagei contains bufadienolides with specific cytotoxic activity on cancer cells without affect to normal cells, suggesting that its consumption could be considered as a potential therapy against cancer

Lung cancer is almost the most common cause of cancer death in the world. Clinically, the conventional therapy to eradicate the cancer cells is chemotherapy but a better drug remains required. In this study, the effects of three bufadienolides, kalantuboside B, kalantuboside A and bryotoxin C, isolated from Kalanchoe tubiflora (Harvey) were evaluated and characterized in CL1-5 highly metastatic human lung cancer cells. Contrary to the apoptosis-promoting activity in other cancer cells, these three bufadienolides did not induce apoptosis in CL1-5 cancer cells. Instead, they activated an autophagy pathway, as indicated by the increase of autophagosomes formation. The induction of autophagy by these three bufadienolides was demonstrated to link to down-regulation of p-mTOR as well as up-regulation of LC3-II, ATG5, ATG7, Beclin-1. Moreover, among these three compounds, kalantuboside B in which a monosaccharide is attached at the bufadienolide aglycon, exhibited a better autophagy induction. Our findings revealed an alternative mechanism of drug action by bufadienolides in lung cancer cells and provided evidence for the possibility of treating highly metastatic human lung cancer through an autophagy pathway.